Previously we identified the highly conserved amino acids Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) on the ecto-domain of gp41 as the epitope of a neutralizing monoclonal antibody (2F5) directed against human immunodeficiency virus type 1. In the present study, the sequence defining the epitope was introduced into the loop of antigenic site B of the influenza virus hemagglutinin. The resulting chimeric virus was able to elicit ELDKWA-specific immunoglobulins G and A in antisera of mice. Moreover, the distantly related human immunodeficiency virus type 1 isolates MN, RF, and IIIB were neutralized by these antisera. These data suggest that this conserved B-cell epitope is a promising candidate for inclusion in a vaccine against AIDS. The results also show that influenza virus can be used to effectively present the antigenic structure of this B-cell epitope.

Cross-neutralizing activity against divergent human immunodeficiency virus type 1 isolates induced by the gp41 sequence ELDKWAS

To obtain direct evidence for a relationship between hemagglutinin (HA) cleavability and the virulence of avian influenza A viruses, we generated a series of HA cleavage mutants from a virulent virus, A/turkey/Ontario/7732/66 (H5N9), by reverse genetics. A transfectant virus containing the wild-type HA with R-R-R-K-K-R at the cleavage site, which was readily cleaved by endogenous proteases in chicken embryo fibroblasts (CEF), was highly virulent in intramuscularly or intranasally/orally inoculated chickens. By contrast, a mutant containing the HA with an avirulent-like sequence (R-E-T-R) at the cleavage site, which was not cleaved by the proteases in CEF, was avirulent in chickens, indicating that a genetic alteration confined to the HA cleavage site can affect cleavability and virulence. Mutant viruses with HA cleavage site sequences of T-R-R-K-K-R or T-T-R-K-K-R were as virulent as viruses with the wild-type HA, whereas a mutant with a two-amino-acid deletion but retention of four consecutive basic residues (R-K-K-R) was as avirulent as a virus with the avirulent-type HA. Interestingly, although a mutant containing an HA with R-R-R-K-T-R, which has reduced cleavability in CEF, was as virulent as viruses with high HA cleavability when given intramuscularly, it was less virulent when given intranasally/orally. We conclude that the degree of HA cleavability in CEF predicts the virulence of avian influenza viruses.

Reverse genetics provides direct evidence for a correlation of hemagglutinin cleavability and virulence of an avian influenza A virus.

IFNs protect from virus infection by inducing an antiviral state and by modulating the immune response. Using mice deficient in multiple aspects of IFN signaling, we found that type I and type II IFN play distinct although complementing roles in the resolution of influenza viral disease. Both types of IFN influenced the profile of cytokines produced by T lymphocytes, with a significant bias toward Th2 differentiation occurring in the absence of responsiveness to either IFN. However, although a Th1 bias produced through inhibition of Th2 differentiation by IFN-gamma was not required to resolve infection, loss of type I IFN responsiveness led to exacerbated disease pathology characterized by granulocytic pulmonary inflammatory infiltrates. Responsiveness to type I IFN did not influence the generation of virus-specific cytotoxic lymphocytes or the rate of viral clearance, but induction of IL-10 and IL-15 in infected lungs through a type I IFN-dependent pathway correlated with a protective response to virus. Combined loss of both IFN pathways led to a severely polarized proinflammatory immune response and exacerbated disease. These results reveal an unexpected role for type I IFN in coordinating the host response to viral infection and controlling inflammation in the absence of a direct effect on virus replication.

Type I IFN Modulates Innate and Specific Antiviral Immunity

Influenza virus polymerase complex is a heterotrimer consisting of polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA). Of these, only PB1, which has been implicated in RNA chain elongation, possesses the four conserved motifs (motifs I, II, III, and IV) and the four invariant amino acids (one in each motif) found among all viral RNA-dependent RNA or RNA-dependent DNA polymerases. We have modified an assay system developed by Huang et al. (T.-J. Huang, P. Palese, and M. Krystal, J. Virol. 64:5669-5673, 1990) to reconstitute the functional polymerase activity in vivo. Using this assay, we have examined the requirement of each of these motifs of PB1 in polymerase activity. We find that each of these invariant amino acids is critical for PB1 activity and that mutation in any one of these residues renders the protein nonfunctional. We also find that in motif III, which contains the SSDD sequence, the signature sequence of influenza virus RNA polymerase, SDD is essentially invariant and cannot accommodate sequences found in other RNA viral polymerases. However, conserved changes in the flanking sequences of SDD can be partially tolerated. These results provide the experimental evidence that influenza virus PB1 possesses a similar polymerase module as has been proposed for other RNA viruses and that the core SDD sequence of influenza virus PB1 represents a sequence variant of the GDN in negative-stranded nonsegmented RNA viruses, GDD in positive-stranded RNA virus and double-stranded RNA viruses, or MDD in retroviruses.

Mutational analysis of the conserved motifs of influenza A virus polymerase basic protein 1.

To investigate the biologic importance of the neuraminidase (NA) stalk of influenza A virus, we generated mutant viruses of A/WSN/33 (H1N1) with stalks of various lengths (0 to 52 amino acids), by using the recently developed reverse genetics system. These mutant viruses, including one that lacked the entire stalk, replicated in tissue culture to the level of the parent virus, whose NA stalk contains 24 amino acid residues. In eggs, however, the length of the stalk was correlated with the efficiency of virus replication: the longer the stalk, the better the replication. This finding indicates that the length of the NA stalk affects the host range of influenza A viruses. The NA stalkless mutant was highly attenuated in mice; none of the animals died even after intranasal inoculation of 10(6) PFU of the virus (the dose of the parent virus required to kill 50% of mice was 10(2.5) PFU). Moreover, the stalkless mutant replicated only in the respiratory organs, whereas the parent virus caused systemic infection in mice. Thus, attenuation of the virus with the deletion of the entire NA stalk raises the possibility of its use as live vaccines.

Biologic importance of neuraminidase stalk length in influenza A virus.

Influenza A and B viruses have not been shown to form reassortants. It had been assumed that the lack of genotypic mixing between influenza virus types reflected differences in polymerase and packaging specificity. In this study, we show that an influenza A virus polymerase transcribes and replicates a chloramphenicol acetyltransferase (CAT) gene flanked by the nontranslated sequences of an influenza B virus gene. Although the transcription level of this CAT gene was several times lower than that of a CAT gene flanked by the homologous nontranslated sequences of an influenza A virus, we proceeded to construct a chimeric type A/B influenza virus. Using recombinant DNA techniques, a chimeric neuraminidase gene was introduced into the genome of influenza A/WSN/33 virus. The hybrid influenza A/B virus gene contained the coding region of the A/WSN neuraminidase and the 3' and 5' nontranslated sequences of the nonstructural gene of influenza B/Lee virus. The resulting chimeric virus formed plaques in Madin-Darby bovine kidney cells but replicated more slowly and achieved lower titers than wild-type influenza A/WSN/33 virus. The chimeric virus was attenuated for mice as indicated by a 400-fold increase in its LD50. Interestingly, the virus was greatly restricted in replication in the upper respiratory tract and partially restricted in the lungs. Animals infected with the transfectant virus were highly resistant to influenza virus challenge. It appears that this chimeric virus has many of the properties desirable for a live attenuated virus vaccine.

https://www.pnas.org/content/pnas/88/12/5177.full.pdf

E K Subbarao

Papers

An influenza A virus containing influenza B virus 5' and 3' noncoding regions on the neuraminidase gene is attenuated in mice.